I need to express a recombinant protein in HEK293 cells. The coding sequnce was cloned into pcDNA6-v5/His A vector without leader peptide. Calcium phosphate or commercial reagent "Turbofect" were used for transient transfection. The highest concentration of protein was 160 ng/ml (attached figure) which was 10 times less then reported in the literature. I used GFP-coding vector to visualise expressing cells. The best results were obtained using calcium phosphate method with chloroqine pretreated cells. 40-50% of cells were positive but most of them remained quite dim.
How can I improve expression efficiency? Will addition of the leader peptide help? If so, which one should I use? Or cloning into a bicistronic vector with resistance gene may solve the problem?
For high level expression you should probably be using HEK 293T cells. 293T cells are derived from 293 but express the SV40 large T antigen. This allows episomal replication for high level vector accumulation in transient expression and also binds SV40 enhancers to increase protein expression. I haven't used HEK for many years, and I have not used pcDNA6, but I remember it made a huge difference to use the right HEK variant at the time. Hope this is of help.
In addition to the 293T cells, you could try different transient transfection reagents to improve transformation efficiency and overall expression levels. I've had pretty good results with Lipofectamine 2000 and 3000-the best results with Lipo3000. I recommend them over Fugene HD, X-fect, X-tremeGene, PolyJet,and PolyPlus jetPRIME; all of which I've used with 293T cells for expression of membrane proteins with pcDNA3.1+ vector.
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